Urea plays a critical role in the urinary concentrating mechanism and therefore in the regulation of water balance. Although the first UT was cloned in 1993 and significant progress was achieved, the molecular mechanisms for UT-A1 regulation in cells are still unclear. We recently identified a number of accessory proteins which might play critical regulatory roles in UT-A1 maturation, trafficking, recycling and degradation. The overall goal of the current application is to investigate how the protein- protein interaction regulates UT-A1 activity in cells. We are particularly interested in a group of small molecular weight proteins, such as caveolin-1 and 14-3-3 proteins. Our preliminary data show that these proteins can physically interact with UT-A1. We choose these three candidates since they represent three kinds of proteins that regulate UT-A1 at different steps with different mechanisms. Understanding these interactions is a critical step in dissecting the processes of UT-A1 intracellular translocation, membrane trafficking, as well as retrieval/degradation that all contribute to the urea transport in kidney under physiological and pathological conditions. Our study will not only provide us new insights to understand UT-A1 cellular regulation (trafficking, recycling and degradation) but also provide new insights in the entire transporter research field. In collaboration with Dr. Haian Fu and the Emory Chemical Biology Drug Discovery Center, our long-term project aim is to screen and develop small-molecule modulators of these proteins (first 14- 3-3) that could affect UT-A1 function in vivo for potential therapeutic interventions for the diseases with total body fluid overload such as hypertension, congestive heart failure, cirrhosis, and nephrotic syndrome. PUBLIC HEALTH RELEVANCE: Sodium and urea are the two major solutes that contribute to the medullary osmolarity gradient in kidney. Urea reabsorption in kidneys is mainly mediated by urea transporter UT-A1 in the inner medullary collecting duct (IMCD) epithelial cells. Although the first urea transporter was cloned in 1993 and significant progress has been achieved, the mechanisms for its regulation are still unclear. The aim of the current application is to explore the molecular mechanisms of how UT-A1 is regulated by accessory proteins. We are particularly interested in a group of small molecular weight proteins (20~30kDa) such as caveolin and 14-3-3. We have preliminary data suggesting that these proteins may play important roles in the UT-A1 urea transporter trafficking, recycling, activation, and degradation. Our long-term goal is to screen and develop small-molecule modulators of these proteins that could affect UT-A1 function in vivo for potential therapeutic interventions for total body fluid overloaded diseases such as hypertension, congestive heart failure, cirrhosis, and nephrotic syndrome.